Can J Diabetes 37 (2013) 375e380

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Canadian Journal of Diabetes journal homepage: www.canadianjournalofdiabetes.com

Original Research

Effects of Aerobic Exercise with or without Metformin on Plasma Incretins in Type 2 Diabetes Saeed Reza Toghi Eshghi MSC, Gordon J. Bell PhD, Normand G. Boulé PhD * Faculty of Physical Education and Recreation, Alberta Diabetes Institute, University of Alberta, Edmonton, Alberta, Canada

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 June 2013 Received in revised form 18 July 2013 Accepted 18 July 2013

Objective: Despite positive effects of incretins on insulin secretion, little is known about the effect of exercise on these hormones. Metformin can affect incretin concentrations and is prescribed to a large proportion of people with diabetes. We, therefore, examined the effects of aerobic exercise and/or metformin on incretin hormones. Methods: Ten participants with type 2 diabetes were recruited for this randomized crossover study. Metformin or placebo was given for 28 days, followed by the alternate treatment for 28 days. On the last 2 days of each condition, participants were assessed during a non-exercise day and a subsequent exercise day. Aerobic exercise took place in the morning and blood samples were taken in the subsequent hours (before and after lunch). Results: Aerobic exercise did not increase total plasma glucagon-like peptide-1 (GLP-1) or glucosedependent insulinotropic polypeptide (GIP) in the pre- or post-lunch periods (all p>0.1). GLP-1 was higher in the pre-lunch (p¼0.016) and post-lunch (p¼0.018) periods of the metformin conditions compared with the placebo. Total plasma GIP was higher in the pre-lunch period (p¼0.05), but not in the post-lunch period (p¼0.95), with metformin compared with placebo. Conclusions: In contrast to our hypothesis, aerobic exercise did not acutely increase total GLP-1 and GIP levels in patients with type 2 diabetes. Metformin, independent of exercise, significantly increased total plasma GLP-1 and GIP concentrations in these patients. Ó 2013 Canadian Diabetes Association

Keywords: glucagon-like peptide-1 glucose-dependent insulinotropic polypeptide metformin physical activity

r é s u m é Mots clés : le glucagon-like peptide-1 polypeptide insulinotrope glucosedépendante metformine l’activité physique

Objectif : En dépit des effets positifs des incrétines sur la sécrétion d’insuline, on en connaît peu sur l’effet de l’exercice sur ces hormones. La metformine, qui est prescrite à une grande proportion de personnes ayant le diabète, peut influer sur les concentrations d’incrétine. Nous avons donc examiné les effets de l’exercice aérobique ou de la metformine, ou des deux, sur les hormones incrétines. Méthodes : Dix (10) participants ayant le diabète de type 2 ont été recrutés pour participer à cette étude croisée à répartition aléatoire. La metformine ou le placébo a été donné pendant 28 jours, et a été suivi de l’autre traitement pendant 28 jours. Les 2 derniers jours de chaque traitement, les participants ont été évalués durant une journée sans exercice et durant une journée comportant de l’exercice. L’exercice aérobique a eu lieu en matinée et les échantillons de sang ont été recueillis au cours des heures subséquentes (avant et après le dîner). Résultats : L’exercice aérobique n’a pas fait augmenter les concentrations plasmatiques totales du GLP-1 (glucagon-like peptide -1) ou du GIP (glucose-dependent insulinotropic polypeptide) durant les périodes précédant et suivant le dîner (tous p > 0,1). Le GLP-1 a été plus élevé durant les périodes précédant le dîner (p ¼ 0,016) et suivant le dîner (p ¼ 0,018) pour ce qui est du traitement à la metformine comparativement à celui du placébo. La concentration plasmatique totale du GIP a été plus élevée durant la période précédant le repas (p ¼ 0,05), mais non durant la période suivant le repas (p ¼ 0,95) pour ce qui est de la metformine comparativement au placébo. Conclusions : Contrairement à notre hypothèse, l’exercice aérobique ne faisait pas augmenter de manière considérable les concentrations totales du GLP-1 et du GIP chez les patients ayant le diabète de type 2. La

* Address for correspondence: Normand G. Boulé, PhD, University of Alberta, Faculty of Physical Education and Recreation, 1-002 Li Ka Shing Centre for Health Research Innovation, Edmonton, Alberta T6G 2E1, Canada. E-mail address: [email protected]. 1499-2671/$ e see front matter Ó 2013 Canadian Diabetes Association http://dx.doi.org/10.1016/j.jcjd.2013.07.030

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metformine, indépendamment de l’exercice, augmentait significativement les concentrations plasmatiques totales du GLP-1 et du GIP chez ces patients. Ó 2013 Canadian Diabetes Association

Introduction Type 2 diabetes is a metabolic disorder characterized by hyperglycemia caused by a combination of insulin resistance and impaired insulin secretion. It has been known for more than 40 years that insulin secretion is greater in response to oral glucose compared with an intravenous glucose load that leads to a similar plasma glucose profile (1). This is known as the incretin effect, and the 2 main incretin hormones are considered to be glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP; also called gastric inhibitory polypeptide) (2,3). Incretin hormones have been shown to be responsible for about 60% of postprandial insulin secretion in healthy subjects (4,5). The secretion of these incretins, especially GLP-1, is impaired in patients with type 2 diabetes (6e8). Pharmacologic interventions targeting these hormones recently were approved in Canada (9). Some of these drugs are classified as GLP-1-receptor agonists (e.g. exenatide and liraglutide) or dipeptidyl peptidase-4 inhibitors (e.g. sitagliptin, saxagliptin and linagliptin) that exert their effects through a dipeptidyl peptidase-4 (DPP-4)-resistant analogue to GLP-1 (10) or by increasing GLP-1 half-life, respectively (11). Compared with the knowledge accumulated on these pharmacologic interventions, very little is known about the effects of exercise on GLP-1 or GIP. This is important because exercise is considered to be a first-line intervention for the prevention and management of type 2 diabetes (12e14). Previous research consistently has shown that exercise can increase incretins in healthy subjects (15,16). However, to our knowledge, only the study of Solomon et al (17) has examined the effect of exercise on incretins. They showed that increased GIP concentrations after 3 months of diet and exercise was significantly related to increased insulin secretion (17). In addition to exercise, metformin also is considered a first-line therapy for type 2 diabetes (18) and this is notable because a large proportion of individuals with diabetes are treated with metformin. Although Canadian data are scarce, the estimated number of metformin prescriptions in the United States has increased from about 38 million in 2006 to more than 48 million in 2010 (top 10 for generic drugs) (19). Yasuda et al (20) showed that a single dose of metformin can significantly increase total GLP-1 in subjects without diabetes (20). Metformin also has been shown to increase plasma concentrations of GLP-1 in rats (20). It is unknown if the combination of metformin and exercise leads to greater incretin responses than after metformin alone. Interestingly, we recently observed that the combination of metformin and aerobic exercise acutely increased glucagon concentrations (21). This increased glucagon could be expected to occur if incretin hormone concentrations were lowered by the combination of metformin and exercise (22e24). Therefore, the objectives of the current study were to examine the effects of aerobic exercise, metformin and their combination on preprandial and postprandial plasma concentrations of GLP-1 and GIP. It was hypothesized that both aerobic exercise and metformin would increase GLP-1 and GIP in people with type 2 diabetes.

Methods Participants Ten volunteers (8 men and 2 postmenopausal women) with type 2 diabetes were recruited for this study, which was approved by the University of Alberta Health Research Ethics Board. The

present study was part of a larger research project for which details have been published previously (21). The GLP-1 and GIP data reported here were not published previously but were measured in the plasma samples taken during the previous study. Briefly, participants met the following eligibility criteria, as previously reported (21): (1) between 30 and 65 years of age; (2) not taking glucose-lowering medication or insulin; (3) no changes in physical activity of more than 1 hour per week over the past 3 months and not planning on changing medication, physical activity or diet over the course of the study; and (4) glycated hemoglobin level of 8% or less, resting blood pressure of 140/90 mm Hg or less, LDL cholesterol level of 3.5 mmol/L or less and total:HDL cholesterol ratio of 5.0 or less. The inclusion criteria and study information provided to participants did not specifically proscribe dietary supplements. Although not specifically required by our inclusion criteria, the women were not on hormone replacement therapy. Study design The study used a 22 factorial design during which each participant was exposed to 4 conditions: (1) metformin and no exercise, (2) metformin and exercise, (3) placebo and no exercise and (4) placebo and exercise. The order of the metformin vs. placebo conditions was assigned randomly by personnel not involved with the study, and allocation was concealed in sealed envelopes until participants completed the study. Participants, study personnel and investigators were blinded to the order of the placebo/metformin conditions. Metformin or placebo was given for 28 days, immediately followed by the alternate condition for 28 days. On the last 2 days of each condition (days 27 and 28), participants returned to the laboratory for a non-exercise and exercise session, respectively. The order of these sessions was not determined randomly and exercise always was performed on day 28 because the acute glucose-lowering effect of exercise may persist for at least 24 hours (25). The experimental design of this study and a portion of the methods have been published previously, but there was no duplication in any of the dependent variables reported in this article (21). Study protocol As previously described (21), a baseline exercise stress test was performed to determine the participants’ peak oxygen uptake (VO2peak) and ventilatory threshold. Participants were given either metformin or placebo pills and were asked to maintain their routine physical activity and dietary habits. Each participant consumed 500 mg of metformin with breakfast during the first week of the intervention followed by a 500 mg increase in each of the subsequent weeks until 1000 mg were consumed with breakfast and supper during week 4 (total, 2000 mg/day). On days 27 and 28 of the metformin and placebo conditions, participants arrived in the laboratory at 8:00 AM after a 12-hour fast. Participants ate a standardized breakfast (549 kcal; 56% carbohydrate, 30% fat and 14% protein) and took their assigned pills. At 10:00 AM, an intravenous catheter was inserted into an antecubital vein kept patent with 0.9% sterile saline. On day 27 of the metformin and placebo conditions, the participants remained at rest for the duration of the testing period. At 10:45 AM on day 28 during both conditions, participants performed a series of exercises that were selected to represent different intensities, modes and energy systems. They began with 20 consecutive maximal leg extensions

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Statistical analyses

Plasma GIP concentration

Analyses were conducted using repeated measures analysis of variance with treatment order added as a between-subject factor. To simplify the interpretation, the testing days were split into

Plasma total GIP concentrations pre-lunch also were unaffected by aerobic exercise (p¼0.38) but were significantly higher on both nonexercise and exercise days with metformin compared with the

Blood sample: Time:

Results Participants had an average body mass index of 28.65.3 kg/m2 and an average age of 586 years. They had relatively wellcontrolled glycemia (glycated hemoglobin, 6.5%0.6%; fasting glucose, 7.30.6 mmol/L). Some participants reported mild to moderate gastrointestinal side effects during the 4-week metformin intervention; but all participants except one (final metformin dosage, 1500 mg/day) were able to tolerate the maximum dosage of 2000 mg/day during the last week of the intervention. Half of the participants started with 28 days of metformin. VO2peak was 30.25.1 mL$kg1$min1 at baseline. The first 15-minute bout was performed at 33.9%5.4% of VO2peak, the second 15-minute bout averaged 67.2%7.3% of VO2peak and the remaining 5-minute bout averaged 79.4%8.8% of VO2peak. Submaximal oxygen consumption was not affected by metformin (p¼0.60). However, the mean respiratory exchange ratio (RER) was lower in the metformin condition (0.960.02 vs. 0.980.02; p¼0.03). Plasma total GLP-1 concentration Plasma total GLP-1 concentrations pre-lunch were unaffected by aerobic exercise (p¼0.48) but were significantly higher in both non-exercise and exercise days with metformin compared with similar days during the placebo conditions (p¼0.02; Figure 2). Plasma concentrations of total GLP-1 remained higher in both metformin conditions during the 1 hour post-lunch period (p¼0.02).

8:00AM

Incretin substudy

Standardized Lunch at 12:00

- 15 min below VT

- 5 min above VT

- Cybex 10:20

- 15 min at 3.5 kph

Exercise or no exercise day Standardized Breakfast + Metformin or Placebo

RANDOMIZATION (28 days of metformin/placebo)

2 periods: pre-lunch and post-lunch. The number of within-factors and levels varied among these periods (e.g. post-lunch was a 222 factorial analysis of variance to examine the main effects of exercise, metformin and time [i.e. 30 and 60 minutes post-lunch], as well as the interactions among these factors). Unless stated otherwise, means  standard deviations are presented. Exploratory analyses also were conducted to examine the association between incretins and insulin, glucagon, the insulin: glucagon ratio as well as glucose. Pearson correlations examined the association among absolute values as well as the incremental effect of the meal. Statistical tests were 2-tailed, and p values of 0.05 or less were considered significant. Statistical analyses were performed with SPSS 21 (SPSS, Inc, Chicago, IL).

Recruitment, Screening & Baseline-Assessment

and flexions on a Cybex II isokinetic dynamometer (Ronkonkoma, NJ, USA). After a 5-minute rest period, the first of 3 aerobic exercise bouts began. Each bout was separated by a 5-minute rest period. During the first aerobic exercise bout, all participants walked at 3.5 km/h at a 0% grade for 15 minutes on a treadmill. This corresponded to the estimated average walking speed for individuals with type 2 diabetes in free-living conditions (26). The second bout also lasted 15 minutes and was completed at a speed and grade equivalent to an exercise intensity below each participant’s measured ventilatory threshold. The third bout was completed at an intensity above their ventilatory threshold and lasted 5 minutes. Figure 1 shows a schematic representation of the study protocol. The earlier-described exercise protocol was developed as part of our original study, which was designed to examine the effect of metformin on various types and intensities of exercise. The mean energy expenditure over the 35 minutes of aerobic exercise was 4.9 metabolic equivalents (21). Consequently, the total energy expenditure from this exercise would be consistent with a 50-minute brisk walk at 3.5 metabolic equivalents (27), which would meet the recent Canadian Diabetes Association minimum recommendation for aerobic exercise if performed 3 times per week (14). Approximately 20 minutes after exercise (at 11:59 AM), a blood sample was taken immediately before the standardized meal (556 kcal; 59% carbohydrate, 22% fat and 19% protein). Participants remained in the laboratory and blood samples were taken at 12:30 and 1:00 PM. Additional blood samples had been taken throughout the day (21); however, only the earlier-described 3 samples were analyzed for GLP-1 and GIP owing to budgetary constraints. These samples were targeted because they were thought to best represent the acute effect of aerobic exercise and meal ingestion. Each blood sample was first transferred into a 10 mL ethylenediaminetetraacetic acid vacutainer tube (Becton, Dickinson and Company, Franklin Lakes, NJ). Tubes were mixed by gentle inversion and centrifuged at 1500  g for 10 minutes, after which the plasma was aliquoted into tubes before cooling and storage in a freezer at 20 C until assays were completed. Total GLP-1 and total GIP were measured according to the manufacturer’s procedures using commercially available enzyme-linked immunosorbent assay kits (EMD Millipore, Billerica, MA). All samples were run in duplicate in each assay. The mean coefficient of variation for GLP-1 and GIP was 3.6% and 2.7%, respectively.

12:00 12:30 13:00

Figure 1. Study protocol. kph, kilometers per hour; VT, ventilatory threshold.

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with the corresponding differences in insulin, glucagon, insulin: glucagon ratio or glucose. No associations were found between differences in incretins and differences in glucose between the metformin and placebo conditions. The only significant correlation that was observed was between the magnitude of the metformininduced changes in GLP-1 and insulin in the postprandial periods from the nonexercise conditions (r¼0.64, p¼0.05). Body weight was 86.918.7 kg at baseline and was similar after the metformin and placebo conditions (mean difference between conditions¼0.00.8 kg). Differences in body weight were not consistently associated with differences in incretins between metformin and control conditions. Discussion

Figure 2. The effect of exercise and metformin on GLP-1 and GIP. Data are reported as meanSEM. Analyses were adjusted for treatment order (i.e. metformin first vs. placebo first). Sample size¼9 or 10. , metformin þ no exercise; -, metformin þ exercise; B, placebo þ no exercise; ,, placebo þ exercise; *, Significant main effect of metformin (p0.05); U, Significant main effect of time (p

Effects of aerobic exercise with or without metformin on plasma incretins in type 2 diabetes.

Despite positive effects of incretins on insulin secretion, little is known about the effect of exercise on these hormones. Metformin can affect incre...
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